Broad-Spectrum Glycoside 3-Dehydrogenase with Distinct Features of Glucose–Methanol–Choline Oxidoreductase for Site-Selective Oxidation of Glycosides and Saccharides

The discovery of genomic glycoside utilization loci (GULs) that enable multistep nonhydrolytic glycoside degradation has revealed previously unexplored oxidative enzymes for glycoside transformation. The pathway-initiating enzyme is a C3-selective oxidoreductase. Here, we characterize the representative GUL-associated enzyme from Rhizobium sp. GIN611 (RhG3DH) and define its evolutionary, mechanistic, and biocatalytic features within the glucose–methanol–choline oxidoreductase superfamily. RhG3DH is functionally and evolutionarily distinct from previously characterized GUL-associated glycoside 3-oxidases, yet shows the closest relationship to flavin adenine dinucleotide-dependent d-fructose dehydrogenase and d-glucose dehydrogenase. Recombinant RhG3DH expressed in E. coli formed a functional complex with its twin-arginine translocation partner. The enzyme is an iron–sulfur flavoprotein, containing a 3Fe–4S cluster, that transfers electrons to cytochrome c, consistent with the organization of the corresponding GUL. In contrast to GUL-associated glycoside 3-oxidases, RhG3DH showed negligible oxidase activity with O2 but efficiently utilized small-molecule electron acceptors. Screening of 31 substrates, and kinetic characterization of a subset thereof, including d-glucose-derived di- and oligosaccharides as well as d-glycosides, revealed an unusually broad substrate scope. RhG3DH tolerated diverse aglycones as well as O- and C-glycosidic linkages, independent of linkage position and α/β-configuration. Despite this breadth, the enzyme displayed strict C3 site selectivity at the nonreducing glycosyl residue and prevented overoxidation even at high conversion. RhG3DH was readily integrated into both an O2-driven laccase cascade reaction, affording 95% conversion of 200 mM sucrose to 3-keto-sucrose, and an electrochemically driven system that achieved 82% conversion of 20 mM sucrose. These results establish RhG3DH as a distinct and broadly useful biocatalyst for selective glycosyl substrate C3 oxidation.